RAL-1 signaling regulates lipid composition in C. elegans

Signaling by the Ral small GTPase is poorly understood in vivo . Caenorhabditis elegans animals with constitutively activated RAL-1 or deficient for the inhibitory RalGAP, HGAP-1 /2, display pale intestines. Staining with Oil Red O detected decreased intestinal lipids in the hgap-1 deletion mutant relative to the wild type. Constitutively activated RAL-1 decreased lipid detected by stimulated Raman scattering (SRS) microscopy, a label-free method of detecting lipid by laser excitation and detection. A signaling-deficient missense mutant for RAL-1 also displayed reduced lipid staining via SRS. We conclude that RAL-1 signaling regulates lipid homeostasis, biosynthesis or storage in live animals.


Figure 1. RAL-1 regulates lipid homeostasis, biosynthesis and/or storage:
A-C) Using an Oil Red O staining protocol we found that lipid storage was decreased in B) hgap-1(∆) relative to (A) wildtype animals.These data are quantitated in (C) as arbitrary units (a.u.) (P < 0.0001; posterior intestine showed).D-H) Stimulated Raman Scattering (SRS) imaging of lipid levels revealed decreased lipid in animals with constitutively active RAL-1.DIC (D, F) and SRS (E,G) imaging of animals with RAL-1 tagged at the N-terminal with mKate2::3xFlag.Wild-type (D, E) were compared to G26V constitutively activated RAL-1 (F, G).Pixel intensity was measured from the SRS images (E, G) in the areas illustrated with red and black circles of 35 μm (D).Background (black circle) was subtracted from the anterior intestine (red circle) in the SRS image to yield a value for lipid content.These data for various experiments were graphed as normalized SRS intensity (H-K).

Description
The Ras small GTPase is the most mutated human oncoprotein: 19% of tumors harbor activating mutations in Ras (Prior, Hood, & Hartley, 2020).Oncogenic Ras utilizes three main direct binding partners, called effectors, that propagate downstream signaling.The Raf>MEK>ERK MAP Kinase pathway and PI3K>PDK>AKT pathway are among the best studied and pharmacologically targeted signaling cascades in all of biology (Cox et al., 2014).In contrast, Ras activation of RalGEF signaling through Ral (RalGEF>Ral) is neglected and poorly understood, despite playing a critical role in Ras-driven tumorigenesis (Apken & Oeckinghaus, 2021).The inhibitory GAP for Ral is also implicated as a tumor suppressor, suggesting that Ral can drive tumorigenesis in the absence of activating mutations in Ras (Beel et al., 2020;Oeckinghaus et al., 2014;Yoshimachi et al., 2021).
Ral (Ras like) is a small GTPase in the Ras family.Ras itself is the founding member of the Ras superfamily of small GTPases.GTP-bound Ral (Ral•GTP) is in the active state and engaging downstream effectors, while GDP-bound Ral (Ral•GDP) is in the inactive state.RalGEF (guanine nucleotide exchange factor) is bound by activated Ras to stimulate nucleotide disassociation of Ral•GDP, upon which free cytosolic GTP spontaneously loads to form Ral•GTP. RalGAP (GTPase activating protein) stimulates the poor intrinsic GTPase activity of Ral to hydrolyze GTP to GDP to yield Ral•GDP, hence inactivating Ral.These general mechanisms for regulating Ral are conserved among the Ras superfamily, including Ras itself and related families Rho, Rab, Arf and Ran (Reiner & Lundquist, 2018;Wennerberg, Rossman, & Der, 2005).
We have also characterized the ral-1(gk628801[R139H]) mutant, which abolishes 2˚ VPC-promoting signal and compromises cell migration events, but which is otherwise superficially wild type (Mardick et al., 2021;Shin et al., 2019;Shin et al., 2018).These signaling deficient animals also display decreased fat content by SRS imaging (Fig. 1I).However, we could not perceive a pale intestine phenotype associated with signaling deficient RAL-1, which suggests that the mechanism of decreased lipid detectable by SRS is distinct from that observed from increased RAL-1 signaling.
We conclude that increase of Ral activation, either through gain of RAL-1 function or loss of inhibitory HGAP, reduces detectable fat content as detected by Oil Red O or SRS.Reduction of RAL-1 signaling activity as assayed in our studies also resulted in decreased lipids, but likely via a distinct mechanism.Though perhaps paradoxical, these observations could reflect differences in mobilization of detectable lipids or metabolism to different molecular species.
The effects of deletion of putative effectors of RAL-1 lead to uninterpretable results.Yet perhaps this is not surprising: these proteins perform an array of complex cellular functions beyond signal transduction.Exo84 and Sec5 are components of the heterooctameric exocyst complex, which performs cell-essential functions in direct exocytosis via the Golgi (Pereira et al., 2023).The exocyst is evolutionarily conserved from yeast to mammals, but unlike metazoans, yeasts do not encode Ral orthologs.In addition to being bound by Ral•GTP, RalBP1 (Ral binding protein 1) functions as a GAP to inhibit Rac and Cdc42 of the Rho family of small GTPases, primarily known for regulating cytoskeletal dynamics.RalBP1 also functions as a non-ABC ATP-dependent transporter with ATP-binding sites, regulates mitochondrial fission/fusion, and associates with EH domain-containing proteins REPS1 and POB1, which function in receptor-mediated endocytosis.(Cornish, Owen, & Mott, 2021).Consequently, all three putative effectors of RAL-1 could be expected to exert complex influence on lipid biosynthesis and/or storage in the animal.Better understanding of these regulatory inputs into storage and metabolism of lipids will require selective missense mutations that uncouple specific functions, partnered with more complex analysis of lipid metabolism.

Methods
Animals were cultured at 20˚C on NGM plates spotted with OP50 E. coli bacteria.All strains are derived from the N2 Bristol wild-type background.
For staining via Oil Red O (ORO), animals were fixed with isopropanol and stained with ORO dye as described (O'Rourke et al., 2009;Wahlby et al., 2014).ORO data were acquired using a Nikon eclipse Ni microscope via epifluorescence or DIC/Nomarski imaging with a Nikon DS-Fi2 color camera.Images were processed using NIS-Elements Advanced software Research, Version 4.40.ORO intensity measurement was performed using Fiji Image J software version 2.1.0/1.53C(NIH).Original color image documents were split into red, green, blue channels (Image → color → split channels).Intensity of red channel was obtained by subtracting blue and green pixel intensity (creating blue + green channel: Process → image calculator.image1: green/ image2: blue/ Operation: average; creating red-only channel: Process → image calculator.image1: red/ image2: result of green + blue channels/Operation: subtract).Average intensity was measured from a circle of 150 pixels in a diameter in the posterior intestine of each animal.
For lipid detection via Stimulated Raman Scattering (SRS), animals were anesthetized with 10 mM tetramisole, mounted on 2% agar pads on glass slides, and subjected to laser stimulation and confocal microscopy image capture as described (Mutlu et al., 2021;Wang et al., 2011).Briefly, an Olympus IX81 inverted laser-scanning confocal microscope optimized for near infrared signal detected signal generated by temporally overlapping Pump and Stokes laser beams and optimized for lipids contained in lipid droplets (Mutlu et al., 2020).Images were processed using Olympus Fluoview 1000 software.